Method of preparing D-luciferin derivatives

ABSTRACT

A method of preparing a 6-substituted D-luciferin ester suitable for use in the determination of pesticides by a bioluminescence method. The 6-substituted D-luciferin esters, such as 6-D-luciferin acetate, are prepared in a liquid solvent containing N-acetyl imidazole with D-luciferin and recovering as the reaction product 6-D-luciferin acetate.

REFERENCE TO PRIOR APPLICATIONS

This application is a divisional application of U.S. Ser. No.08/007,572, filed Jan. 22, 1993, now abandoned, which application was adivisional application of U.S. Ser. No. 07/818,782, filed Jan. 9, 1992,now U.S. Pat. No. 5,283,180, issued Feb. 1, 1994, which patent is acontinuation-in-part of U.S. Ser. No. 07/556,952, filed Jul. 19, 1990,now U.S. Pat. No. 5,200,311 issued Apr. 6, 1993.

The parent application concerns a radio assay test method employing C¹⁴radiolabeled materials which includes adding insect brain tissue, suchas bee or housefly brain tissue homogenate, to a test sample containingan organophosphate or carbamate pesticide which interacts with the brainhomogenate. The admixture is incubated and then a radiolabeledsubstrate, for example, a C¹⁴ substrate, is added to interact with theremaining sites in the incubated insect brain homogenate and the mixtureincubated. The resulting incubated test sample, brain homogenate andradiolabeled substrate is then separated to provide a liquid fractionsample to which is added a scintillation material. The radioactivity ofthe separated fraction is then determined by counts per minute andcompared with a standard or control to determine the concentration ofthe pesticide in the test sample.

BACKGROUND OF THE INVENTION

Different kinds of organisms (arthropods, avians, mammals) are sensitiveto pesticides. Pesticides are generally classified as herbicides,fungicides and insecticides. Pesticides interact with their nervous andenzymatic systems. Such toxicants may bind to binders (ion channels)located on the nerve cells, or to enzymes located around them andelsewhere. Pesticides also interact with various protective mechanisms,such as degrading enzymes and non-specific binders.

The brain and the nerve system of insects in general were the target forinsecticides since their early development (1940s) for pest control inagriculture and human health. Pesticides that target the insect brain asthe site of action usually display rapid action and require low dosagefor good control.

Since the early 1970s, attempts have been made to use in vitro brainpreparation to study the mode of action of pesticides. Only limitedstudies were reported on using this method for monitoring pesticides.Lack of stability and sensitivity as compared with the traditional gaschromatography (GC) or mass spectrometry made this early attemptuseless. The use of color reaction requires high enzyme and substrateconcentration to monitor brain preparation activity and the results wereinsensitive assays. The enzymes used were taken through laboriouspurifications, and it was found that only a limited spectrum ofpesticides could be detected. Apparently, the source and the purifiedpreparation were of limited sensitivity and for only a few pesticideswhich made it unsuitable as a monitoring and screening assay.

In recent years, bioluminescence or chemiluminescence has been used todetect extremely small quantities of material. Assays using thesesignals have been developed to measure substrates, such as ATP or NADH,and enzymes, such as alkaline phosphatase. Advantages to usingbioluminescence as an assay system is the sensitivity of the reactionand the speed in which that reaction can be measured. For example, thebioluminescence reaction with the enzyme luciferase catalyzes thereaction between luciferin and ATP to produce light in milliseconds.

It is desirable to test for the concentration of pesticides in variousmaterials, such as soil and water for health and safety purposes. Inparticular, it is desirable to provide an effective test kit and methodto determine the concentration level of organophosphate and carbamatepesticides at low levels, such as below 50 ppb and even as low as 5 ppb.Specific organophosphate insecticides may be tested employingantibodies, but these have limited use as broad spectrum screeningmethods. Herbicides may be tested on a specific basis by chromogenicenzyme-based test methods, but such tests do not provide accurateresults at low concentration levels and are susceptible to colorinterpretation.

Therefore, a new, accurate, effective test kit and method for thedetermination of pesticides, such as organophosphate and carbamatepesticides, are desirable.

SUMMARY OF THE INVENTION

The invention relates to a test kit and method for the determination ofpesticides. In particular, the invention concerns a chemiluminescence orbioluminescence test method and test kit for the determination oforganophosphate and carbamate-type pesticides at levels below about 50ppb.

A test kit and method has been developed for the rapid, generally 10 to15 minutes, and sensitive, less than 50 ppb or lower, method for themultiple detection of organophosphate and carbamate pesticides in water,soil, food (e.g. meat, fish, fruits and vegetables) and other materials.The test method employs an insect brain preparation having a mixture ofreceptors or enzymes with sites that interact with the pesticides andparticularly that react or interact with organophosphate and carbamatepesticides. The pesticides alter or cause a significant reduction in thebrain activities of the insect brain preparation which are inverselycorrelated with the amount of the pesticide present in the test sample.The activity is measured by employing tracer analogs or substrates whichupon exposure to the insect brain preparation are altered chemicallyand/or physically and the changes monitored by a light emissionreaction. The activities can be chemical as in an enzymatic reaction orphysical as in receptor binding.

For example, in one embodiment in an enzymatic reaction, the hydrolysisof a luciferin derivative to luciferin by a brain preparation has beenfound to be extremely sensitive to organophosphates and carbamates. Theluciferin liberated by the reaction is oxidized by the enzyme luciferaseand adenosine triphosphate (ATP), an energy donor, and emits lightmeasured as bioluminescence or chemiluminescence. The bioluminescence orchemiluminescence emitted is measured and monitored at a low level andhigh speed employing a luminometer or scintillation counter. The assayis carried out in three simple steps of a short incubation period, e.g.2 to 10 minutes, of the test sample and the brain preparation; additionof a substrate tracer or pesticide analog tracer, such as a luciferinderivative with additional incubation, e.g. 2 to 5 minutes; and themeasurement directly or after a separation step to obtain liquidfraction for light emission by activation with the enzyme(luciferin-luciferase). The general assay preparation is illustrated inTable 1.

Therefore, a test method has been found for the determination ofpesticides which are sensitive to insect brain preparation andparticularly organophosphate and carbamate pesticides. The test methodcomprises incubating a mixture of a test sample and an insect brainpreparation; adding to the incubated mixture a D luciferin derivative,such as, but not limited to: D luciferin acetate, a novel compound whosehydrolysis is inhibited in the presence of the pesticide; incubating thetest sample, brain preparation and D luciferin derivative mixture toliberate luciferin; admixing a portion of the incubated D luciferinderivative-containing admixture with ATP and luciferase as a reactionmixture to provide an oxidized luciferin (oxyluciferin) and emittedluminescence; measuring the emitted luminescence, for example, with aluminometer; and determining the concentration of the pesticide in thetest sample by comparison of the emitted, measured luminescence of astandard or of a control sample.

The test kit for the determination of pesticides comprises incombination an insect brain preparation which is sensitive to thepesticide, e.g. bee brain homogenate, a D luciferin derivative which isinhibited in hydrolysis in the presence of the pesticide, such as Dluciferin acetate; the enzyme luciferase and ATP to form a reactionmixture when added together to the incubated test sample, brainpreparation and D luciferin derivative. The test kit may include thosestandard articles of laboratory equipment and chemicals, like buffers,needed to carry out and measure the results to include, but not belimited to: incubation dishes or plates and an incubation water bath,etc.; buffers; a luminometer to measure emitted luminescence; a standardcontrol chart or graph of luminescence vs. pesticide concentration forcomparison and determination of the pesticide concentration; andseparating equipment, such a chromatographic column or ultrafiltrationmembrane to obtain a liquid fraction of the incubated mixture.

A wide variety of insect brain tissue material may be used in the testmethod of the invention, such as the crude, stabilized brain tissue,particularly the homogenate of arthropods, for example, but not limitedto: bees; beetles; aphids; mosquitoes; silkworms; mites; blow flies; andhouseflies (Musca domestica) alone or in combination.

Brain preparations from various sources have differences in specificityfor pesticides. Therefore, it is important to obtain one or more brainpreparations which will react with a broad spectrum of pesticides formonitoring purposes. Insects as the target for pesticides are one of thebest sources for brain preparations. Specificity and sensitivity canvary from one insect to another. Bees were chosen as a preferred choicefor their known sensitivity to a variety of insecticides. It was foundthat bee brain preparation was approximately 2 to 4 logs more sensitivein the bioluminescence assay system than any previously reported assaysystem.

One novel D luciferin derivative suitable for use in the pesticide testmethod comprises 6-acetyl D luciferin or luciferin acetate, a novelcompound, as its homolog. Suitable luciferin derivatives are those6-substituted D luciferin compounds which in the presence of insectbrain preparation react to cleave the substituted ester group at the6-position of the D luciferin derivative to provide D luciferin forfurther reaction with ATP and luciferase to produce luminescence whichcan be measured. The luciferin derivative 6-acetyl D luciferin isprepared by reacting D luciferin with excess acetyl imidazole in asolvent, e.g. water-solvent solution which acetylates D luciferin undermild conditions with about 100% efficiency to provide the 6-acetyl Dluciferin.

It has been found that both organophosphate and carbamate pesticidesinhibit the hydrolysis of D acetyl luciferin by insect brainpreparations. There are significant advantages in the use of a Dluciferin derivative, like D luciferin acetate, as a substrate in thetest method. There is an unusual, unexpected, high specificity of thebee or silkworm brain preparations toward D luciferin acetate and onlysmall quantities of the D luciferin acetate are required for the assay.For example, the concentration of D luciferin acetate in the incubationmixture may be as low as 1×10⁻¹¹ moles or less and the assay sensitivityfor luciferin is 1 to 5×10⁻¹³ moles. The assay time is about 10 minutein total. While D luciferin acetate has been found to be a preferred Dluciferin derivative for use in the test method, other D luciferinderivatives with similar reactions may be employed. Those luciferinderivatives disclosed in the publication of the Journal of ClinicalChemistry and Chemical Biochemistry entitled "Synthesis andCharacterization of Luciferin Derivatives for Use inBioluminescence-Enhanced Enzyme Immunoassay New Ultrasensitive DetectionSystems for Enzyme Immunoassay", Miska et al, J. Clin Chem Clin Biochem25(1) 1987, pp. 23-30, are not suitable for use in the test method andinclude specifically D-luciferin methyl ester,D-luciferyl-L-phenylalanine, D luciferyl-L-Na-arginine,D-luciferin-O-sulphate and D-luciferin-O-phosphate.

To test the ability of the assay system to monitor for the presence ofpesticides in food, six apples from different sources were tested. Ofthe six apples, four apples were positive and two apples were negative.The four positive apples were obtained from local orchards, while thenegative apples were purchased from local supermarkets. Local water wasalso tested and was found marginal positive. Table 3 gives detectionlevels expected in water for 15 different organophosphate and carbamatepesticides.

Test Procedure Used for Organophosphate and Carbamate Pesticides

1. Sample is preincubated with a predetermined quantity (dilution 1:200)of brain preparation for 5 minutes.

2. Luciferin acetate (200 pmoles) is added to the incubation mixture foradditional 5 minutes.

3. A portion of this incubation mixture (100 μl) is withdrawn and addedto the luciferase and ATP reaction mixture (1 ml).

4. Bioluminescence reading for 2-5 seconds is monitored.

5. The reading of the pesticide sample is compared to the control sampleto quantitate the percent inhibition of the sample (see Tables 3, 4, 5and 6).

The invention will be described for the purposes of illustration only inconnection with certain embodiments; however, it is recognized thatthose persons skilled in the art may make various improvements,additions, changes and modifications to the illustrated embodiments allfalling within the spirit and scope of the invention.

DESCRIPTION OF THE EMBODIMENTS A. Synthesis of luciferin acetate(6-acetyl D luciferin)

Dissolve 2 mg sodium D-luciferin (purified from firefly) into 2 mlwater, or dissolve 2 mg synthetic D-luciferin into 2 ml methanol asstock 3.3 mM luciferin solutions in an amber vial. Take 25 μl of eitherstock solution and add it to 1 ml distilled water in an ambermicrocentrifuge tube to get an 82.5 μM solution of luciferin. Dissolve60 mg of N-acetylimidazole into 1.0 ml acetone to get a 545 mM solutionof N-acetylimidazole. Add 30 μl of 545 mM solution of N-acetylimidazoleto the 82.5 μM aqueous solution of luciferin. Mix several times andmonitor the decrease of bioluminescence as the reaction continues tocompletion. The reaction is complete when bioluminescence can no longerbe observed when using the above reaction mixture as a source forluciferin. Keep solution on ice. The concentration of N-acetylimidazolein this reaction mixture is 16.35 mM or approximately 200 times theconcentration of luciferin.

Notes: D-Luciferin from firefly was purchased from Sigma. SyntheticD-luciferin was purchased from Boehringer Mannheim. Also, a D-luciferinis available from Bio-Orbit. Luciferin stock solution from Sigma wasdissolved in water while D-luciferin stock solution from Boehringer orBio-Orbit was dissolved in methanol. N-acetylimidazole was purchasedfrom Sigma. The reaction described above was performed in 1 ml and 1concentration range, but it could be scaled up using a larger volumesize. Other derivatives of luciferin, such as luciferin phosphate,luciferin sulfate, luciferin arginine have been described in theliterature, but to our knowledge, luciferin acetate has not beendescribed, nor to our knowledge has it been used in a coupled reactionwith brain extract to monitor pesticides.

B. Preparation of Insect Brain Extract

Insects are stored from at -20° C. or below. Insect heads are collectedby dissection with a scalpel and placed in a 50 ml beaker containingapproximately 15 ml ice cold 0.07 M phosphate buffer, pH 7.0, containing1 mM EDTA (ethylenediaminetetra acetic acid) and 1 μM phenylthiourea.The insect heads are gently homogenized at low speed in a TekmarTissumizer. Aliquots of this crude extract are transferred tomicrocentrifuge tubes and centrifuged. The supernatant is retained andapplied to a sephadex G-25 column equilibrated with 0.07 M phosphatebuffer, pH 7.0, containing 1 mM EDTA and 1 μM phenylthiourea. Fractionsare collected every 2 minutes and monitored for esterase activity usingluciferin acetate as a substrate and measuring bioluminescence.Fractions with esterase activity are pooled and used as the insect headextract for use in the pesticide assay.

Notes: Brain extracts have been prepared primarily from honey bees, butalso has been extracted from silkworm and blowflies. Proteinconcentration of the bee head extract is between 2 to 5 mg/ml. For theassay, 5 to 10 μl is used per assay.

C. Bioluminescence Reaction

1. Reaction buffer for bioluminescence: Weigh out 4.48 g tricine, 0.6 gmagnesium sulfate, 0.146 g EDTA, 100 mg bovine serum albumin, and 77 mgdithiothreitol into 600 ml water. Adjust pH to 7.8 with 10% sodiumhydroxide and add distilled water to 1 liter.

2. Luciferase preparation: For luciferase (1 mg) from firefly purchasedfrom Boehringer Mannheim dissolve in 1 ml 0.5 M Tris-acetate buffer, pH7.5, and let stand 30 minutes. Portion out 40 μl of this solution intoglass test tubes and freeze at -20° C. Redissolve frozen stock with 1 mlbioluminescence buffer. For luciferase purchased from Bio-Orbit take 3mg and dissolve in 1 ml bioluminescence buffer. Each luciferase solutionis kept on ice. For each bioluminescent assay, a 30 μl aliquot fromeither luciferase solution described above is used. For the Boehringerluciferase, approximately 1 μg enzyme is used for each assay.

3. ATP Preparation: ATP (adenosine 5'-triphosphate) is preweighed fromSigma and contains 1 mg ATP and 40 mg magnesium sulfate. To this vialadd 10 ml water and 100 μl 1.0 M HCl and vortex. ATP solution is kept onice. The stock solution is 1.8 mM and for each bioluminescent assay, 30μl is used for a final concentration of 54 μM.

4. Luciferin Acetate: Take 100 μl 82.5 μM solution of luciferin acetateprepared as described above in section A and 900 μl distilled water andadd to amber microcentrifuge tube. This solution is 8.25 μM. Forpesticide assays, a 25 μl portion of the 8.25 μM solution is added to 2ml 0.07 M phosphate buffer, pH 7.0, to give a 0.1 μM concentration ofluciferin acetate. A 100 μl portion of this solution is withdrawn andincubated with 1 ml of the bioluminescent buffer. Final concentration isapproximately 10 nM luciferin acetate or 10 pmoles luciferin acetate.

5. Bioluminescence Measurement: A 1 ml portion of the bioluminescentbuffer is added to a 13×100 mm test tube. A 100 μl portion of theluciferin acetate solution (0.1 μM) is withdrawn and added. Then 30 μlaliquots of the luciferase and ATP solutions are added to the test tube.The test tube is vortexed and the bioluminescence measured after 2 to 5seconds using a luminometer. The bioluminescence reading with luciferinacetate will be negative. If luciferin is measured at 10 pmoles then thereading in the luminometer will be approximately 100,000. If theconcentration of any of the reagents described above is increased, thenthe light reading will be increased. Although the present concentrationsgive acceptable values, the concentration of the reagents may be alteredto give optimal performance. The above liquid reagents may also beimmobilized in individual tablet form to provide stability andconsistency to the reagents, such as in compressed tablet form withinert cellulosic fillers.

D. Assay of Pesticides Using Bioluminescence

1. To 13×100 mm test tubes add 2 ml 0.07 M phosphate buffer, pH 7.0. Add25 μl positive pesticide control (organophosphate and/or carbamatepesticide), 25 μl water to negative control tube and 25 μl of testsample to another tube. If test sample contains solvent other thanwater, then use this solvent as the negative control tube.

Note: Although 25 μl is indicated above, the volume size of sample couldbe less or greater as long as brain activity is not affected by thesolvent.

2. Add insect brain extract (5 or 10 μl depending on activity) to the 2ml incubation mixture in step 1, vortex and incubate at 35° C. intemperature block for 10 minutes. Use timer with alarm to monitor time.

3. At 10 minutes, add 25 μl 8.25 μM luciferin acetate solution to 2 mlincubation mixture in step 2. Reset timer to zero and restart timer.

4. At 5 minutes, take 100 μl portion from each sample. Use separatepipette tip for each sample and add the 100 μl portion to separate13×100 mm test tubes containing 1 ml of bioluminescence buffer.

Note: In this reaction, the sample is withdrawn after 5 minutes. Sincethe reaction is kinetic, the concentration of luciferin formed in thereaction will increase with time. Therefore, the time an aliquot can bewithdrawn can vary as long as it is in the linear section of the curve,and aliquots from samples are withdrawn at the same time.

5. Add 30 μl ATP solution followed by 30 μl luciferase solution to oneof the assay tubes in step 4. Vortex and measure bioluminescence for 2to 5 seconds. Record value and proceed to the next tubes as above.

6. Zero control bioluminescence reaction will be uninhibited bypesticide and will have a high bioluminescence reading. In the positivesamples the esterolitic activity of the bee extract will be inhibitedand less luciferin will form giving a low bioluminescent reading. Dividesample reading or positive control by zero control and multiply to 100to get percent inhibition.

Note: The source for some of the chemicals used has been set forth;however, other suppliers can supply these chemicals and therefore, theprocedure is not reliant on any one source for a particular reagentexcept for the preparation of luciferin acetate.

Tables 5-11 represent test data showing the increase in bioluminescenceas a function respectively of time, luciferase, ATP, luciferin acetateand bee head brain preparation in the test assay.

                  TABLE 1                                                         ______________________________________                                        Schematic of Pesticide Assay                                                  ______________________________________                                         ##STR1##                                                                      ##STR2##                                                                      ##STR3##                                                                      ##STR4##                                                                     ______________________________________                                         Legend                                                                        .sup.a) for example, insect bee brain homogenate                              .sup.b) organophosphate and carbamate pesticides                              .sup.c) for example, D luciferin acetate                                      .sup.d) luminometer                                                      

                  TABLE 2                                                         ______________________________________                                        PREPARATION OF D LUCIFERIN ESTER                                              ______________________________________                                         ##STR5##                                                                      ##STR6##                                                                      ##STR7##                                                                      ##STR8##                                                                     ______________________________________                                         LEGEND                                                                        "R" IS AN ALKYL GROUP OR PHENYL GROUP, e.g. C.sub.1 -C.sub.6             

                  TABLE 3                                                         ______________________________________                                        Charm Pesticide Assay for Organophosphate and Carbamate                       Inhibition of Bee Brain Activity for Ac-luciferin                                              I.sub.50.sup.a)                                              Pesticide        (ppb)                                                        ______________________________________                                        Carbamates                                                                    Methomyl         4                                                            Propoxur         10                                                           Carbofuran       8                                                            Bendocarb        12                                                           Organophosphates                                                              Mevinphos        2                                                            Ethion           2                                                            Chlorpyros       1                                                            Phorate          2                                                            Malathion        6                                                            Oxydemeton-methyl                                                                              1                                                            Disulfoton       5                                                            Methyl parathion 1                                                            DDVP             0.004                                                        Naled            0.05                                                         Diazinon         15                                                           ______________________________________                                         .sup.a) I.sub.50 is the concentration of inhibitor which gives a 50%          decrease in enzymatic activity as measured by bioluminescence.           

                  TABLE 4                                                         ______________________________________                                        Inhibition of Silkworm Brain Activities by                                    Various Organophosphate Pesticides at 25 PPB                                                   %                                                            Pesticide        Inhibition                                                   ______________________________________                                        Phorate          50.5                                                         Naled            98.0                                                         Methyl parathion 0                                                            Ethion           77.0                                                         Oxydemeton-methyl                                                                              0                                                            Diazinon         0                                                            DDVP             100.0                                                        Disulfoton       25                                                           Mevinphos        50                                                           ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Inhibition of Bee Brain Extract by Organophosphates                           As a Function of Pesticide Concentration                                      ______________________________________                                        Methyl Parathion                                                                          Phorate       Chlorpyrifos                                               %                 %              %                                     ppb    activity ppb      activity                                                                             ppb     activity                              ______________________________________                                        0      100      0        100    0       100                                   0.625  62       1.25     70     0.625   68                                    1.25   40       2.5      29     1.25    34                                    2.5    23       3.125    23     2.50    24                                    5.0    15       6.25     8      3.125   13                                                    12.5     3      6.25    10                                    ______________________________________                                        Ethion      Diazinon      Oxydemeton-methyl                                          %                 %              %                                     ppb    activity ppb      activity                                                                             ppb     activity                              ______________________________________                                        0      100      0        100    0       100                                   0.3125 84       25       40     0.3125  90                                    0.625  71       50       45     0.625   43                                    1.25   65       100      28     1.25    47                                    2.5    37                       2.5     37                                                                    25.0    21                                    ______________________________________                                        Disulfoton  Mevinphos     Malathion                                                  %                 %              %                                     ppb    activity ppb      activity                                                                             ppb     activity                              ______________________________________                                        0      100      0        100    0        100                                  3.125  66       0.3125   64     6.25    53                                    6.25   47       0.625    52     12.5    50                                    12.5   35       1.25     48     25.0    27                                    25.0   19       2.5      24     50.0    27                                                    3.125    12                                                                   6.25     9                                                    ______________________________________                                        DDVP               Naled                                                      ppb      % activity    ppb     % activity                                     ______________________________________                                        0        100           0       100                                            0.0013   74            0.031   50                                             0.0023   49            0.625   29                                             0.0028   60            0.125   30                                             0.0057   52            0.250   25                                             0.011    25                                                                   ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        Inhibition of Bee Brain Extract by Carbamates                                 As a Function of Pesticide Concentration                                      ______________________________________                                        Bendiocarb   Methomyl      Carbofuran                                         ppb   % activity ppb     % activity                                                                            ppb   % activity                             ______________________________________                                        0     100        0       100     0     100                                    12.5  50         3.125   45      6.25  59                                     25    26         6.25    16      12.5  41                                     50    18         12.5    20      25    32                                     100   11         25.0    12      50    22                                     ______________________________________                                               Propoxur                                                                      ppb   % activity                                                       ______________________________________                                               0     100                                                                     3.1   62                                                                      12.5  43                                                                      25.0  44                                                                      62.5  29                                                                      125.0 27                                                               ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        Increase In Bioluminescence as a Function of Time                             Using Pesticide Assay in Control Reaction                                     Time (min)    Light reading                                                   ______________________________________                                        0             0                                                               0.75          390                                                             1.75          1230                                                            2.75          3030                                                            3.75          3510                                                            5.0           5550                                                            7.0           9360                                                            9.0           11460                                                           11.0          13200                                                           14.0          20640                                                           16.0          27300                                                           ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Increase in Bioluminescence as a Function of Luciferase                       Using the Pesticide Assay in Control Reaction                                 Luciferase (μg)                                                                           light reading                                                  ______________________________________                                        0              0                                                              0.17           390                                                            0.34           1200                                                           0.5            1470                                                           0.67           4830                                                           0.83           7740                                                           1.0            8580                                                           1.17           17280                                                          1.34           20700                                                          1.51           31868                                                          1.68           40140                                                          ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        Increase in Bioluminescence as a Function of ATP                              using the Pesticide Assay in Control Reaction                                 ATP (μM)   Light Reading                                                   ______________________________________                                        0             0                                                               0.9           390                                                             1.8           1289                                                            3.6           4110                                                            5.4           8220                                                            9.0           20760                                                           10.8          24060                                                           ______________________________________                                    

                  TABLE 10                                                        ______________________________________                                        Increase in Bioluminescence as a Function of Luciferin Acetate                Concentration Using the Pesticide Assay in Control Reaction                   Luciferin     Light                                                           Acetate (μM)                                                                             Reading (at 3 min)                                              ______________________________________                                        0             0                                                               0.2           8520                                                            0.4           15120                                                           0.8           61680                                                           1.6           180240                                                          3.2           467280                                                          6.4           724080                                                          ______________________________________                                    

                  TABLE 11                                                        ______________________________________                                        Increase in Bioluminescence as a Function of Bee Brain Extract                Concentration Using Pesticide Assay in Control Reaction                              Bee      Light                                                                Extract (μl)                                                                        Reading                                                       ______________________________________                                               0          0                                                                  10       1920                                                                 20       6484                                                                 40       22260                                                                80       28880                                                         ______________________________________                                    

What is claimed is:
 1. A method of preparing a 6-substituted D-luciferinester compound, which method consists essentially of:a) reacting in aliquid solvent a substituted N-acylated imidazole compound withD-luciferin; and b) recovering a solution of 6-substituted D-luciferinester compound.
 2. The method of claim 1 wherein the liquid solventcomprises a water solvent mixture.
 3. The method of claim 1 wherein theliquid solvent comprises water.
 4. The method of claim 1 wherein thesubstituted N-acylated imidazole compound comprises N-acetylimidazole inan acetone-water solvent.
 5. The method of claim 1 which includesmonitoring the decrease in bioluminescence of the luciferin as a measureof the completeness of the reaction.
 6. The method of claim 1 whereinthe substituted group of the N-acylated imidazole compound is selectedfrom the group consisting of: alkyl, benzyl and phenyl radicals.
 7. Themethod of claim 1 wherein the liquid solvent comprises methanol, and theD-luciferin is a synthetic D-luciferin.
 8. The method of claim 1 whereinthe N-acylated imidazole compound is N-acetyl imidazole.
 9. The methodof claim 8 wherein the substituted N-acylated imidazole compound is usedin stoichiometric excess.
 10. A method of preparing a 6-substitutedacetyl D-luciferin ester compound, which method consists essentiallyof:a) reacting in a liquid solvent a stoichiometric excess of N-acetylimidazole with D-luciferin to provide 6-acetyl-D-luciferin estercompound and imidazole; and b) recovering a liquid solvent solution ofthe 6-acetyl-D-luciferin ester compound for use in a bioluminescencetest method.